This invention relates generally to the fields of inorganic and nucleotide chemistry. More specifically, the invention relates to sterically demanding nucleotide intercalating compounds and methods for detecting base pair mismatches in nucleic acid sequences.
DNA base-pair mismatches arise during the course of genetic recombination and replication as a consequence of enzymatic errors or DNA damage. In the cell, complex systems exist to recognize, remove, and repair these mistakes to preserve the integrity of the genetic code. In certain diseases, particularly cancer, these repair systems fail and mismatches persist in a diseased cell""s DNA. The design of molecules and systems to recognize site specific mismatches in DNA is an attractive experimental goal both for genetic screening and for the design of new chemotherapeutics. Existing strategies include assays using isolated mismatch recognition proteins, hybridization of oligonucleotide-fluorescent probe conjugates, electrophoretic/DNA chip methods, and differential chemical cleavage with reagents assaying for base access ability either in solution or the solid phase. None of these methods are ideal for detection of mismatches in the laboratory, and no strategies exist for using the presence of base-pair mismatches as a way of selectively treating disease.
Accordingly, there is a need for compositions and methods useful for recognizing site specific base-pair mismatches in polynucleotide duplexes.
The present invention is based on the discovery of a new class of sterically demanding metallo-intercalators. These compounds intercalate between bases in a duplex polynucleotide, but only where the bases are not fully complementary, for example, where there is a base-pair mismatch. The compounds are sufficiently sterically demanding that intercalation between bases in fully complementary duplexes does not occur to a significant degree. These mismatch intercalators are useful for detecting DNA and RNA defects, for diagnosing disorders characterized by the presence or increase in DNA and/or RNA defects, and for treating such disorders. The compounds of the invention are useful for SNP (single-nucleotide polymorphism) discovery. The compounds are also useful as carriers of cytotoxic agents targeting cells having deficient repair mechanisms (e.g., in various cancer cells).
A new class of compounds of the formula M(R1)(R2)(R3)3+ and derivatives thereof is described herein. Invention compounds intercalate only between nucleotide bases in the presence of a base-pair mismatch, and are therefore useful for detecting single base-pair mismatches. Further, the compounds are capable of catalyzing photolytic cleavage of nucleic acids at relatively long wavelengths, and under normal sunlight.
In accordance with the present invention, there are provided compounds of the formula M(R1)(R2)(R3)3+ and derivatives thereof.
In another aspect of the invention there are provided compositions comprising two complementary oligonucleotide strands having a base-pair mismatch, and compounds of the formula M(R1)(R2)(R3)3+.
In a further aspect of the invention, there are provided methods for determining the presence of a base-pair mismatch in a polynucleotide duplex, methods for diagnosing genetic disorders characterized by the presence or increased presence of DNA and/or RNA defects (for example base-pair mismatches), methods for treating disorders characterized by the presence or increased presence of DNA and/or RNA defects, and methods for screening agents for their ability to damage nucleic acid duplexes.
In a still further aspect of the invention, the compounds are useful for labeling or indicating base-pair mismatches. In yet another aspect of the invention, the compounds are capable of catalyzing cleavage of a polynucleotide duplex having a base-pair mismatch. In a further aspect of the invention, the compounds are useful for diagnosing and/or treating disorders characterized by the presence of base-pair mismatches in nucleic acid duplexes.